The circadian clock serves as a temporal filter to time gene expression, cell metabolism, physiology and behavior to the most critical moments in the day, thus contributing to the organism's adaptation to a changing environment. While the molecular mechanisms that generate and sustain rhythmicity at the cellular level are well understood, it is less clear how this information is further structured to control specific behavioral outputs. Rhythmic release of pigment dispersing factor (PDF) has been proposed to propagate the (time of day) information from core pacemaker (PDF reactive) cells to downstream targets underlying rhythmic locomotor activity. Indeed, such circadian changes in PDF intensity represent the only known mechanism through which the PDF circuit could communicate with its output. Recently, we reported a novel circadian phenomenon involving extensive remodeling in the axonal terminals of the PDF circuit which display higher complexity during the day and significantly lower complexity at nighttime. Thus, clock-controlled structural plasticity could be a candidate mechanism contributing to the transmission of the information downstream of pacemaker cells. The aim of the present proposal is to extend this initial observation and characterize the structure of the PDF circuit by time- lapse imaging in cultured brains. This approach, coupled with immunohistochemistry, will shed light on whether synaptogenesis and synapse elimination is concomitantly taking place. In addition, the molecular mechanisms responsible for such substantial remodeling will be explored employing novel genetic tools that allow spatial and temporal control of gene expression. Gaining understanding in a model system like Drosophila most likely will impact the current thinking of how the clock controls sleep/activity cycles in mammals. PUBLIC HEALTH RELEVANCE: Circadian systems evolved as a mechanism that allows organisms to adapt to the environmental changes in light and dark which occur as a consequence of the rotation of the Earth. Because of its unique repertoire of genetic tools, Drosophila is a well established model for the study of the circadian clock. Although the biochemical components underlying the molecular oscillations have been characterized in detail, the mechanisms used by clock neurons to convey information to the downstream pathways remain elusive. We have recently discovered a novel form of network plasticity whereby a circuit that is central to rhythmic rest-activity cycles undergoes substantial circadian remodeling and might be critical for clock control of behavior. The aim of this proposal is to extend the initial observations and characterize the molecular mechanisms underlying this form of structural plasticity. Thus, this project will shed light into some novel mechanisms by which neuronal circadian clocks regulate physiology and behavior.